The field of invention is Vibrio cholerae vaccines. After more than 100 years of research on cholera, there remains a need for an effective cholera vaccine. There have been six pandemics of this disease caused by strains of V. cholera belonging to the "Classical" biotype. The etiological agents of the current (seventh) pandemic belong to the "El Tor" biotype (Finkelstein, Crit. Rev. Microbiol 2:553-623, 1973, Wachsmuth et al., The Lancet 337:1097-1098, 1991). Recently the seventh pandemic has extended to a new locale, that of South America. Beginning in January of 1991, an epidemic of cholera resulted in greater than 250,000 cases and over 2,000 deaths in Peru, Ecuador, Columbia, and Chile. Before this epidemic it was estimated that over 200,000 cases of cholera occurred per year mainly in India, Bangladesh, Africa and Western Asia (Tacket et al., Cholera Vaccines. In Vaccines: New Approaches to Immunological Problems, Ellis, R. W., editor, Butterworth-Heinemann, Boston, 1992).
In November of 1992, an antigenically distinct, non-01 form of V. cholerae emerged in India and Bangladesh and within eight months caused an estimated 500,000 cases and 6,000 deaths. The pandemic potential of this new strain, designated serogroup 0139 synonym "Bengal", seems assured and is a new cause of concern throughout the developing world. These recent experiences underline the need for effective cholera vaccines against disease due to both El Tor 01 and Bengal 0139 serotypes of V. cholerae.
Because natural infection by and recovery from cholera induces immunity lasting at least 3 years (Tacket et al., Supra; Levine et al., J. Infect. Dis. 143:818-820, 1981; Cash et al., J. Infect. Dis. 130:325-333, 1974), much effort has been made to produce live, attenuated cholera vaccines that when administered orally would mimic the disease in its immunization properties but would not cause adverse symptoms or reactions in the immunized individual (i.e., display low reactogenicity). Vaccines of this type involve deletion mutations that inactivate the gene encoding the A subunit of cholera toxin, a protein which is responsible for most of the diarrhea seen in this disease (Mekalanos et al., Proc. Natl. Acad. Sci. U.S.A. 79:151-155, 1982; Mekalanos et al., Nature 306:551-557, 1983; Kaper et al., Nature 308:655-658, 1984; Kaper et al., Biotechnology 2:345, 1984; Pierce et al., Infect. Immun. 55:477-481, 1987; Taylor et al., Vaccine 6:151-154, 1988; Levine et al., Infn. Immun. 56:161-167, 1988; Herrington et al. J. Exper. Med. 168:1487-1492, 1988; Levine et al., Lancet ii:467-470, 1988; Kaper et al., Res. Microbiol. 141:901-906, 1990; Pearson et al., Res. Microbiol. 141:893-899, 1990). See also Mekalanos, U.S. Pat. Nos. 5,098,998 and 4,882,278, and Kaper et al., U.S. Pat. No. 4,935,364, hereby incorporated by reference. While both oral, killed whole cell vaccines and several live, attenuated cholera vaccine have been developed, the most promising of these provide little protection against the El Tor biotype of V. cholerae and probably no protection against the 0139 serotype. The major issues associated with cholera vaccines are safety, stability and their degree of antigenicity.
With regard to the toxin genes of V. cholerae, the genetic diversity among toxigenic and non-toxigenic strains has been examined by Chen et al. (1991, Epidemiol. Infect. 107:225). Mekalanos (1983, Cell 35:253) reports on the duplication and amplification of V. cholerae toxin genes, and Miller et al. (1984, Proc. Natl. Acad. Sci. U.S.A. 81:3471) discusses transcriptional regulation of the toxin genes. Other V. cholerae genes whose products may play a role in the pathogenicity of this organism include the toxin-coregulated pilus genes (Shaw et al., 1990, Infect. Immun. 58:3042; Sharma et al., 1989, Vaccine, 7:451; Sun et al., 1990, J. Infect. Dis. 161:1231; Hall et al., 1991, Infect. Immun. 59:2508; Taylor et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:2833), and the gene encoding the intestinal colonalization factor (Taylor et al., 1988, Vaccine 6:151).